The present invention relates generally to methods and materials for the detection of ketones and aldehydes in fluid (liquid or vapor) samples. The invention is particularly directed to the quantitative determination of ketone and aldehyde concentrations in physiological fluids including blood, urine and breath samples. The invention further relates to methods and materials for monitoring the effects of diet, exercise and diabetic conditions through the quantitative measurement of breath acetone levels.
It is known that "ketone bodies" by which term is generally meant acetone, acetoacetic acid and .beta.-hydroxybutyric acid, tend to accumulate in the blood stream during periods of relative or absolute carbohydrate deprivation due to the breakdown of storage triglycerides. The process through which overproduction of ketone bodies occurs is not well defined but is related to increased oxidation of long chain fatty acids by the liver. Specifically, acetoacetic acid and .beta.-hydroxybutyric acid are formed by the liver as intermediates during the oxidation of fatty acid molecules by acetoacetyl coenzyme A. Acetone is formed from the spontaneous decarboxylation of acetoacetic acid. Under normal conditions the intermediate products are further degraded to carbon dioxide and water and the ketone products do not appear at significant concentrations in the bloodstream. Nevertheless, certain metabolic and disease states interfere with the normal degradation of these intermediates which then accumulate in the bloodstream as a result.
The quantitative measurement of ketone concentrations in blood serum is important because of the relationship between elevated serum ketone levels and clinical conditions such as diabetes, disorders of the digestive organs, renal insufficiency, uremia and malignant carcinoma. In the course of these disorders, ketone bodies pass into the blood stream and a state of metabolic acidosis (ketosis) occurs. Monitoring for the onset of ketosis is of particular importance in the maintenance of diabetics because the occurrence of ketosis may indicate the need for modification of insulin dosage or other disease management.
The concentration and identity of various ketone and aldehyde components present in the serum may be determined by direct chemical or chromatographic analysis. While such direct analysis provides the most accurate determination of serum ketone and aldehyde concentrations it suffers from numerous deficiencies including the requirement that blood be drawn to provide serum for analysis. Moreover, the analysis must be carried out promptly due to decomposition of acetoacetic acid to acetone during storage. In addition, the analysis of blood serum for ketones and aldehydes by chemical means requires the use of various reagents and procedures which can be complex and inconvenient for consumer use. Further, the use of certain chromatographic techniques such as gas chromatography is often impractical for consumer and many types of professional use.
As a consequence of the limitations of measuring serum ketone levels directly, a large body of art has developed directed to the testing of urine for the presence of ketone bodies. It is known that the concentration of ketone bodies in urine bears an imperfect relationship to serum ketone concentrations. While urine ketone concentrations depend on numerous factors and are not always directly proportional to serum ketone concentrations, testing of urine for ketones is a simple and relatively inexpensive means of monitoring serum ketone concentrations. Such methods are in widespread use by diabetics in both home and clinical settings.
A number of test devices and methods for the determination of urine ketone concentrations are known to the art. Some assays utilize the reaction of acetone with salicylaldehyde in alkaline solution to give the deeply colored orange to red compound salicylalacetone. Any acetoacetic acid in such solutions is converted by the alkali to acetone which further contributes to the color reaction.
Kamlet, U.S. Pat. No. 2,283,262 discloses compositions for the detection of acetone and acetoacetic acid in solutions such as urine. The materials comprise a dry mixture of a member of the group consisting of the alkali metal and alkali-earth metal bisulfite addition products of salicylaldehyde and a member of the group consisting of the alkali metal and alkali-earth metal oxides and hydroxides.
Many assays take advantage of the "Legal" method which utilizes the reaction of a carbonyl group containing compound such as a ketone or an aldehyde with a nitroprusside (nitroferricyanide) salt in the presence of an amine to form a colored complex. While acetone will react, albeit slowly, with nitroprusside under aqueous conditions, the reaction of acetoacetic acid is some 100 to 200 times faster with the result that "Legal" reactions under aqueous conditions whether detecting "acetone," "acetone bodies" or "ketone bodies" primarily detect acetoacetic acid. The color reaction is believed to occur as a result of a coupling reaction through the nitroso group of the nitroprusside with the analyte to form an intermediate which then complexes with the amine to produce a color characteristic of the specific amine. In forming the complex, the trivalent iron of the nitroprusside is reduced to its divalent state. The color complex, however, is unstable because nitroprusside decomposes rapidly in alkaline solutions. Further, nitroprusside salts are subject to decomposition in the presence of moisture and high pH. Frequently during storage, a brown decomposition product is formed which can interfere with sensitive detection during assays. These limitations have led to numerous attempts to stabilize the color complex by utilizing mixtures of nitroprussides and amines or amino acids in combination with a variety of buffers, metal salts, organic salts, organic stabilizers and polymers. Numerous combinations of reagents have been shown to be suitable for detection of a variety of ketone bodies in liquid samples although the analyte predominantly detected in physiological fluids is acetoacetic acid.
Fortune, U.S. Pat. No. 2,186,902 discloses the use of soluble nitroprusside chromogens in the presence of ammonia and soluble carbonates for the detection of what was termed "acetone" (actually acetoacetic acid) in urine samples. Varying colorations are observable for the quantitative determination of "acetone" levels.
Galat, U.S. Pat. No. 2,362,478 discloses a solid reagent for the detection of "acetone" (actually acetoacetic acid) in liquid samples. The reagent comprises a dry mixture of a powdered anhydrous soluble nitroprusside, granular anhydrous soluble nitroprusside and granular anhydrous ammonium sulfate. The reagent signals the presence of "acetone" by producing a color reaction when a drop of sample is added thereto.
Free, U.S. Pat. No. 2,509,140 discloses improvements on the materials of Fortune comprising solid dry formulations which may be in the form of tablets for the detection of "acetone bodies" or "ketone bodies" in liquids. The materials comprise a nitroprusside salt, glycine and an alkaline salt.
Nicholls, et al., U.S. Pat. No. 2,577,978 discloses improvements on the dry formulations of Free for the detection of "acetone bodies" or "ketone bodies" in bodily fluids. Such compositions comprise alkali metal nitroprussides and alkali metal glycinates combined with sugars such as lactose, dextrose and sucrose.
While many assay devices of the prior art utilize dry tablets or powders in performing an assay, other assay devices utilize adsorbant carriers upon which some or all of the reagents have been dried. The adsorbant carriers may be in the form of strips which can be immersed in a sample of the liquid to be analyzed with the color reaction taking place in solution on the carrier. These assay devices, like those utilizing tablets or powders, suffer from decomposition of the nitroprusside indicator. In addition, indicator materials which are merely adsorbed onto the adsorbant carriers tend to suffer from diffusion of reagents away from the strip which affects the strength of the color signals. Further, the strips exhibit a certain amount of "bleeding" of color product in the aqueous environment which limits the stability of the color indicator signal of the reacted device.
Magers, et al., U.S. Pat. No. 4,147,514 discloses test strips for the detection of ketone bodies such as acetoacetic acid in bodily fluids utilizing a solution comprising nitroprusside in combination with at least one inorganic metal salt where the metal is selected from the group of magnesium and calcium. The solution optionally comprises at least one primary amine combined therewith. Test strips are dipped in the solution and are dried. They may be immersed in fluid samples and the occurrence of a color reaction observed.
U.K. Patent No. 1,012,542 discloses methods for the detection of ketone bodies in bodily fluids wherein alkaline components, in an aqueous solution are impregnated onto a carrier to which, sodium nitroprusside salt in an organic carrier also containing large amounts of an organic film-forming polymer is later applied. The carrier material is said to be very stable and is used for the detection of ketone bodies (acetoacetic acid) in liquid samples.
U.K. Patent No. 1,369,138 discloses improved methods for the detection of ketones in bodily fluids wherein an absorbant carrier is first impregnated with a solution consisting of an amino acid, tetrasodium ethylenediamine-tetraacetate buffer and water which is then dried. The carrier is then impregnated with a solution of sodium nitroprusside in dimethyl formaldehyde and optionally an alcohol containing one to four carbons and is dried.
Smeby, U.S. Pat. No. 2,990,253 discloses a device for the detection of ketone bodies in fluid samples comprising a bibulous carrier onto which nitroprusside is first applied in an aqueous acidic media and to which is subsequently applied a non-aqueous solution of organic bases such as amines or amino alcohols to achieve the alkalinity necessary for the assay reaction.
Mast, et al., U.S. Pat. No. 3,212,855 discloses an improved method for the production of a "dipstick" device for the detection of ketone bodies in fluids in which a bibulous carrier is first impregnated with an aqueous solution comprising an alkaline buffer and a water soluble amino acid. The carrier is then dried and impregnated with a solution in an organic solvent comprising an alkali metal nitroprusside and an organic film producing polymer.
Takasaka, Japanese Patent Application No. 1980-45270 discloses methods for the detection of ketones in body fluids utilizing test strips impregnated with alkali metal salts of nitroprusside and yttrium metal salts. The strips indicate a color reaction in acidic pHs in the presence of acetoacetic acid.
Federal Republic of Germany Patent No. 3,029,865 discloses improved test strips for the detection of ketones in bodily fluids comprising absorbant carriers impregnated with sodium nitroprusside, a water-soluble amino acid, an alkaline buffer compound and phosphoric acid trimorpholide as a stabilizer.
Kikuchi, Japanese Patent Application No. 1982-10208 discloses test strips for the detection of ketones in bodily fluids which are produced by immersion of absorbant carrier material in a solution comprising an amino acid, sodium triphosphate and sodium hydroxide and distilled water. The carrier strips are then dried and are immersed in a solution comprising a nitroprusside salt dissolved in dimethylformamide. They are then dried again and are ready for use.
Hirsch, U.S. Pat. No. 4,097,240 discloses a process for the production of dipstick devices for the detection of ketones in fluids such as urine. The process comprises the impregnation of an absorbant carrier with sodium nitroprusside, an alkaline buffer substance and a water soluble amino acid. The carrier is first impregnated with an aqueous solution of amino acid and tetrasodium ethylenediamine tetraacetate buffer and dried. It is then impregnated with a solution of sodium nitroprusside in a solvent mixture consisting of methanol and an organic solvent miscible with methanol such as a linear or branched aliphatic alcohol with two to six carbon atoms.
Habenstein, U.S. Pat. No. 4,184,850 discloses a dipstick device for the detection of ketone bodies in fluids comprising an absorbant carrier medium impregnated with sodium nitroprusside, a water-soluble lower amino acid, an alkaline buffer substance, and at least one organic acid which serves to form a stabilizing environment around the nitroprusside salt.
Kohl, U.S. Pat. No. 4,405,721 discloses devices for the detection of ketone bodies in bodily fluids comprising a carrier impregnated with a buffer, an amino acid, sodium nitroprusside and a heterocyclic stabilizing compound.
Tabb, et al., U.S. Pat. No. 4,440,724 discloses devices for the detection of ketone bodies in bodily fluids and methods for their preparation. The devices may be constructed according to steps comprising; impregnating a carrier with an aqueous solution of a soluble nitroprusside chromogen, drying the carrier, impregnating the carrier with an aqueous solution including a metal salt, a primary amine, TAPS (N-Tris (hydroxymethyl) 3-aminopropane sulfonic acid) and TRIS (tris-hydroxymethyl aminomethane) and drying the carrier, the pH of the finished test device being no greater than 7.0.
Of interest to the present application is the disclosure of Ogawa, et al., U.S. Pat. No. 3,880,590 which discloses a dipstick device for the semiquantitative detection of acetoacetic acid in liquids such as urine. The Ogawa, et al. strip is said to be incapable of detecting other ketone bodies, such as acetone and .beta.-hydroxybutyric acid. The device comprises an absorbant material, a nitroprusside salt and a heavy metal salt such as nickel or ferric chloride. The absorbant materials include silica gel paper, diethylaminoethyl (DEAE) cellulose paper and amino ethyl cellulose paper with which the nitroprusside salt is associated. The absorbant strips are impregnated with a solution of a nitroprusside salt and a heavy metal salt in water or organic solvents including dimethyl formamide, dimethyl sulfonate methanol and ethanol or mixtures thereof. Solvents disclosed to be useful in forming the devices include dimethylformamide, dimethylsulfoxide, methanol and ethanol and mixtures thereof. According to one example, dimethyl formamide solution is used to impregnate DEAE cellulose paper along with nickel chloride and sodium nitroprusside. The strips were dried and later used to detect the presence of acetoacetic acid in urine. It is disclosed that the impregnating solution itself may be useful for the detection of ketone bodies but that the dried test strips are preferred in view of preservation, stability and handling considerations.
While references variously refer to the use of nitroprusside and amine compositions for the detection of "acetone", "acetone bodies" and "ketone bodies", the assays primarily detect acetoacetic acid and are generally incapable of distinguishing between reaction products formed from reaction of acetone and reaction products formed from reaction of other ketone bodies including acetoacetic acid. Other assays, such as those of Ogawa, et al. are disclosed to be incapable of detecting acetone at all. While numerous advances have been made with respect to "Legal" assays for the detection of ketones and aldehydes, such assays are still limited by the instability of nitroprusside at pHs greater than 7. Finally, such assays still measure only the concentrations of ketone bodies in urine and fail to necessarily provide accurate measurements of ketone bodies present in the blood serum.
It is well known in the art that breath samples may be assayed for the presence of acetone in order to determine serum acetone levels. Acetone is a relatively volatile compound having a partition coefficient of approximately 330. It readily diffuses from the blood into the alveolar air of the lungs according to an equilibrium relationship. As a consequence of this equilibrium state, concentrations of acetone in alveolar air are directly proportional to those in the blood and measurements of acetone in alveolar air can be used to determine the concentration of acetone in the serum. Crofford, et al., Trans. Amer. Clin. Climatol. Assoc. 88, 128 (1977). Crofford, et al. also discloses the use of head space analysis to determine the ketone concentration of liquid samples.
Current methods for the measurement of breath acetone levels include the use of gas chromatography. Rooth, et al., The Lancet, 1102 (1966) discloses the use of a gas chromatograph capable of detecting acetone at concentrations of 2 to 4 nM of air with 18 nM being the concentration for breath of normal individuals. Subjects breathe directly into the device and the acetone peak is read after 40 seconds. Reichard, et al., J. Clin. Invest. 63, 619 (1979) discloses gas chromatographic techniques for the determination of breath acetone concentrations wherein breath samples are collected through the use of a calibrated suction flask into which the test subject breathes through a glass inlet tube. These methods and the instruments required for their use are complicated and expensive and tend to be impractical for use by consumers.
Other methods for the measurement of breath acetone levels involve the use of mass spectrographic equipment. Krotosynski, J. Chrom. Sci., 15, 239 (1977) discloses the use of mass spectrographic equipment in evaluating the ketone content of alveolar air. Twelve ketone components of breath were identified with acetone comprising the major component. Mass spectrographic methods suffer from the same limitations, however, as relate to gas chromatographic techniques.
Methods utilizing color reactions for the detection of acetone in liquid or air have also been reported in the art. Greenberg, et al., J. Biol. Chem. Vol. 154-155, 177 (1944) discloses methods for the determination of acetone levels apart from those of other ketone bodies in solution. The methods involve reaction of acetone and other ketones with 2,4-dinitrophenylhydrazine, to form hydrazone products which may then be separated and isolated owing to differing solubilities.
Peden, J. Lab. Clin. Med. 63,332 (1964) discloses improvements over the methods of Greenberg, et al. utilizing salicylaldehyde as a coloring reagent. According to this method, .beta.-hydroxybutyric acid is converted to acetone by oxidation with the amount of acetone formed measured by reaction with salicylaldehyde. Preformed acetone and acetoacetic acid are removed prior to the conversion of the .beta.-hydroxybutyric acid by heating in the presence of acid. While these methods are useful for the determination of acetone concentrations apart from those of other ketone bodies they are complex and time consuming.
These various colorimetric methods known for detection of acetone in biological fluids are complex, time consuming and necessitate the use of a spectrophotometer or color charts. Moreover, the methods often require the use of high concentrations of alkali or acids. Alternative methods for the detection of acetone often require the use of complex and expensive apparatus. There thus continues to exist a need for methods for the quantitative determination of fluid acetone concentrations which are simple, accurate, inexpensive and do not require the use of high concentrations of hazardous reagents.
There exists a desire for methods for the measurement of the rate of fat catabolism. It is a particular problem that many individuals undergoing diets are unable to determine their rate of fat-loss because of daily variation in their body fluid content. Significantly, it is known that early in a diet individuals lose high proportions of fluid as compared to fat. Later in their diets, when individuals may still be catabolizing fat at a constant rate they may cease to lose fluids at the previous high rate or may, if only temporarily, regain fluid weight. The experience of hitting a plateau in weight loss or even regaining weight is psychologically damaging and weakens the subject's resolve to continue with the diet often with the effect that the subject discontinues the diet.
Recently, a method has been disclosed for the determination of daily rate of fat loss. Wynn, et al., Lancet, 482 (1985) discloses that daily fat-loss may be calculated by subtracting daily fluid and protein mass changes from daily weight changes. Changes in body water are estimated from the sum of external sodium and potassium balances and protein mass changes are calculated from nitrogen balances. Such a method is complex and time consuming thus making it inconvenient for the consumer.
One set of methods for measuring body fat is by quantitating total body water (TBW). A number of methods are available for determining TBW. These include isotopic dilution procedures using deuturiated water, tritiated water and .sup.18 O-labelled water. Urine, blood serum or saliva samples are collected after a 2 to 4 hour equilibration. The fluid samples are then vacuum sublimed and the concentration of tracer in the sublimate is determined by mass spectrometer, gas chromatography, or infrared or nuclear magnetic resonance spectroscopy. Body composition can also be measured by a bioelectrical impedence method using a body composition analyzer.
Hydrostatic weighing method is a well known method wherein the subject is completely submerged in a tank of water and the body fat is calculated by taking into account the average density of fat and the amount of water displaced. This method is inconvenient and is still not completely accurate because assumptions must be made relating to nonfat density, lung capacity and other factors. Another method for calculating the percentage of body fat utilizes skin calipers to measure the thickness of fat deposited directly beneath the skin. Pincers are used to measure the thickness of folds of skin and fat at various locations on the body. The results of these measurements are compared with standardized tables to arrive at a figure for percentage of body fat. This method, while more convenient than the use of hydrostatic weighing is less accurate. All methods for determination of body fat content suffer from the fact that they do not reveal the rate of fat loss but only the fat content of the body at a particular time. Because means for determining body fat content are of limited accuracy, means for the determination of the rate of fat loss are similarly limited. Nevertheless it is desired that a simple and convenient method be developed for the determination of the rate of fat-loss wherein such a method is capable of distinguishing weight loss due to loss of fat as opposed to weight loss from the elimination of bodily fluids.
Of interest to the present invention are observations that ketosis occurs in non-diabetic individuals undergoing weight loss through diet, fasting or exercise. Freund, Metabolism 14, 985-990 (1965) observes that breath acetone concentration increases on "fasting." It is disclosed that breath acetone concentrations increased gradually from the end of the first day of the fast to approximately 50 hours into the fast at which time the concentration rose sharply in a linear fashion and reached a plateau on the fourth day. The acetone concentration of the plateau was approximately 300 .mu.g/liter (5,000 nM) a hundred-fold increase over the normal value of 3 .mu.g/liter (50 nM). When, instead of fasting, the subject was placed on a "ketogenic" diet with a minimum of 92% of calories derived from fat, the subject suffered a lesser degree of ketosis wherein the plateau had an acetone concentration of approximately 150 .mu.g/liter (2,500 nM).
Rooth, et al., The Lancet, 1102-1105 (1966) discloses studies relating to the breath acetone concentrations of a number of obese and diabetic subjects. When the caloric intake of three non-diabetic obese subjects was reduced, their breath acetone concentrations as measured by a gas chromatograph increased approximately three-fold. On fasting, the subjects' breath acetone concentrations increased to one hundred times normal. Within 16 hours after a heavy meal the subjects' breath acetone concentrations dropped almost to normal. In a study of obese diabetic patients, the authors disclosed evidence that those obese patients who had lost weight in the last three months had higher breath acetone concentrations than those patients who had gained weight.
Walther, et al., Acta Biol. Med. Germ. 22, 117-121 (1969) discloses the results of a study on the effects of continued exercise of a well-trained cyclist. The authors disclose that breath acetone concentration, increases prior to, during and after the cessation of the physical load and reached a maximum 15 to 20 minutes after cessation of the physical load. Breath acetone concentrations approach a normal level one to two hours after the cessation of the physical load. It is suggested that the increased production of acetone is due to the increased utilization of plasma free fatty acids in liver and reduced utilization in peripheral tissue.
More recent studies have shown a correlation between fasting in normal and obese patients and increased blood acetone levels. Rooth, et al., Acta Med. Scand. 187, 455-463 (1970); Goschke, et al., Res. Exp. Med. 165, 233-244 (1975); and Reichard et al., J. Clin. Invest. 63, 619-626 (1979) all show the development of ketosis in both overweight and normal individuals during fasting. Rooth, et al., (1970) suggests the use of breath ketone measurements as a motivational tool to enforce against dietary cheating. The studies disclose that development of ketosis is slower in overweight than in normal weight individuals. Reichard, et al., discloses that there is a better correlation between breath acetone and plasma ketone concentrations than between urine ketone and plasma ketone concentrations. In addition, Rooth, et al., (1970) discloses that certain urine ketone tests which detect the presence of acetoacetic acid are not entirely reliable because some individuals do not excrete acetoacetic acid in the urine despite increased blood serum concentrations.
Crofford, et al., (1977) discloses the use of breath acetone monitoring for monitoring of diabetic conditions and as a motivational tool in following patients on long-term weight reduction programs. Such monitoring is said to be particularly effective as normalization of the breath acetone is disclosed to occur upon significant dietary indiscretion. Patients' breath samples were monitored using a gas chromatograph and it is suggested that patients be instructed to restrict their caloric input to that which will maintain breath acetone concentrations of approximately 500 nM. It is further suggested that if breath acetone is controlled at this level and the proper balance of carbohydrate, protein and fat are maintained in the diet that weight loss will occur at a rate of approximately one-half pound per week.